Effect of silicon nanoparticle-based biochar on wheat growth, antioxidants and nutrients concentration under salinity stress

Globally, salinity is an important abiotic stress in agriculture. It induced oxidative stress and nutritional imbalance in plants, resulting in poor crop productivity. Applying silicon (Si) can improve the uptake of macronutrients. On the other hand, using biochar as a soil amendment can also decrease salinity stress due to its high porosity, cation exchange capacity, and water-holding capacity. That’s why the current experiment was conducted with novelty to explore the impact of silicon nanoparticle-based biochar (Si-BC) on wheat cultivated on salt-affected soil. There were 3 levels of Si-BC, i.e., control (0), 1% Si-BC1, and 2.5% Si-BC2 applied in 3 replicates under 0 and 200 mM NaCl following a completely randomized design. Results showed that treatment 2.5% Si-BC2 performed significantly better for the enhancement in shoot and root length, shoot and root fresh weight, shoot and root dry weight, number of leaves, number of tillers, number of spikelets, spike length, spike fresh and dry weight compared to control under no stress and salinity stress (200 mM NaCl). A significant enhancement in chlorophyll a (~ 18%), chlorophyll b (~ 22%), total chlorophyll (~ 20%), carotenoid (~ 60%), relative water contents (~ 58%) also signified the effectiveness of treatment 2.5% Si-BC2 than control under 200 mM NaCl. In conclusion, treatment 2.5% Si-BC2 can potentially mitigate the salinity stress in wheat by regulating antioxidants and improving N, K concentration, and gas exchange attributes while decreasing Na and Cl concentration and electrolyte leakage. More investigations at the field level are recommended for the declaration of treatment 2.5% Si-BC2 as the best amendment for alleviating salinity stress in different crops under variable climatic conditions.


Experimental site and soil collection
A pot experiment was conducted at the Islamia University of Bahawalpur, Botany Department, during the 2021-2022 wheat growing season.The soil was collected from the departmental nursery for the cultivation of wheat.The pre-experimental soil characteristics include pHs (8.45), ECe (3.24 dS/m), nitrogen (0.0025%), phosphorus (7.17 µg/g), exchangeable K (85 µg/g), and soil organic matter (0.35%).

Synthesis of Silicon nanoparticle-based Biochar
Sugarcane press mud biochar synthesis was reported in a previous study.Biochar was produced using the technique described by 16 .Sugarcane press mud was air-dried before being pyrolyzed for 4 h at 450 °C in a muffle furnace 17 .Using the technique of 18 , the produced biochar was sampled, crushed, and sieved with a 0.250 mm strainer.Since the BC sample was collected from our nearby sugar industry (Ashraf Sugar Mill Ltd.BWP) and sugarcane-grown lands in our environment are silicon enriched, the traces of SiO 2 dominate over other nutrients, i.e., P, Mg, Ca, etc.

Characterization of Si-BC
Characterization of biochar was performed as previously described 19 .However, the surface morphology of sieved biochar (sBC) was observed using a ZEISS SEM microscope get on with a 15 kV accelerating voltage.To perform quantitative/elemental analysis, an energy dispersive X-ray (EDX) equipped with the SEM was employed on the sBC powder sample.This sample's X-ray diffraction (XRD) was carried out through Bruker-D8 Advance X-ray Diffractometer with Cu-Kα radiation (λ = 1.54 Å), set to 35 mA current with 40 kV applied potential.To analyze the structure, the XRD instrument scanned the sBC sample at room temperature in 20°-80°.To examine various functional groups present in sBC powder, a Tensor: 27 (Bruker) FTIR spectrometer was run in the frequency range 400-4000 cm −1 , including a few mg of the powder sample mixed with KBr chemical to form a pellet for the analysis.

Seeds collection
For experimental purposes, seeds of Triticum aestivum cultivar (ASS-2011) were purchased from a certified seed dealer in Bahawalpur.The seeds were initially screened out manually.After that, the seeds were sterilized using a 0.1% mercuric chloride solution for 5 min.Afterward, the seeds were rinsed with sterilized water 3 times to eliminate the residual effects of mercuric chloride.

Pots preparation and seeds sowing
Plastic pots were used to conduct the experiment.The dimensions of pots were 20 cm in diameter and 30 cm deep.Each pot was filled with 6 kg of soil.A total of 15 seeds were sown initially in each pot.When seeds were germinated, thinning was performed to maintain 5 seedlings per pot.

Fertilizer
N, P, and K were applied at the rate of 52, 46, and 25 kg/acre (0.39, 0.34, and 0.19 g/pot) for providing macronutrients.For nitrogen, urea fertilizer was used.However, for P and K, single superphosphate and potassium sulfate were used.Urea was applied in 3 splits, while P and K were applied in a single split at the time of pot preparation.

Irrigation
The moisture contents of each pot were maintained at 65% field capacity regularly by using the moisture meter (YIERYI 4 in 1; Shenzhen, Guangdong Province, China).

Harvesting and data collection
Plants were harvested after 75 days of sowing.The data regarding morphological attributes was collected soon after harvesting (Figure S2).For the fresh weight of samples, analytical balance was used.However, sample drying was done in an oven at 65 °C for 24 h to collect the dry weight data of samples.

Chlorophyll estimation
For analysis of chlorophyll contents in the fresh leaves of wheat, samples were ground in 80% acetone.After that, filtration and absorbance were taken at 663 and 645 nm wavelengths on UV spectrophotometer 20 .The final values for chlorophyll a, b, and total were computed using the eq.The photosyn Q meter version 2.0 was used to determine the characteristics of chlorophyll fluorescence.

Leaf relative water content
Initially, 0.5 g of fresh-weight leaf sample was selected for analysis.The sample's turgid weight (TW) was then measured after immersing it in 100 ml of distilled water for 4 h, and the weight was recorded accordingly.The sample underwent oven drying at 70 °C for 48 h to obtain its dry weight (DW) 21 .The final values were obtained using the eq.

Antioxidant enzyme
Nitro blue tetrazolium was used as per standard protocol for assessing the SOD activity by taking absorbance at 560 nm 22 .The enzymatic breakdown of hydrogen peroxide (H 2 O 2 ) for CAT activity was determined by taking absorbance at 240 nm 23 .For APX activity, the reaction between ascorbic acid and H 2 O 2 was noted at 290 nm wavelength 24 .However, malondialdehyde (MDA) was quantified via thiobarbituric acid method 25 .

Electrolyte leakage
Leaf sections weighing one gram each were placed into individual test tubes containing 20 ml of deionized water.These test tubes were then maintained at a steady temperature of 25 °C for 24 h, after which the electrical conductivity of the solution (EC1) was assessed using a calibrated EC meter.Subsequently, the test tubes were heated at 120 °C for 20 min in a water bath, followed by the recording of the second electrical conductivity measurement (EC2) 26 .

Total soluble sugar and total soluble protein estimation
The soluble protein concentration was evaluated using the Bradford assay 27 .Fresh roots and shoots weighing 0.5 g each were homogenized in 10 mL of phosphate buffer with a pH of 7.8 and then centrifuged for 20 min at 10,000 revolutions per minute (rpm).Following centrifugation, 0.1 mL of the protein extraction was mixed with 0.9 mL of tris-HCl buffer and 5 mL of G-250 Coomassie reagent, and the mixture was left at room temperature for 2 min.Absorbance was measured at 595 nm using distilled water as the blank.The protein concentrations were determined using a bovine serum albumin (BSA) standard curve.0.1 mL of plant extract was produced in 25 mL test tubes and used to estimate the soluble sugars using the 28 method.Each tube was heated for 10 min in a boiling bath and then filled with 6 mL of anthrone reagent.After filling, the contents were solidified at room temperature for 10 min.The tubes were then incubated for an additional 20 min following solidification.Subsequently, the optical spectrum was read at 625 nm using a spectrophotometer.

Ions estimation
To analyze Mn, Fe, Cu, Zn, K, and Na, samples were digested using a diacid mixture (nitric and perchloric acid in 3:1 ratio) 29 .The digested sample was run on a flame photometer to determine K and Na 30

Statistical analysis
Standard statistical procedure was followed for the statistical analysis of the data 32 .Two factorial ANOVA was applied for the determination of significance.Each treatment was compared using the Tukey Test at p ≤ 0.05 using OriginPro software 33 .A paired comparison was applied to make the graphs on OriginPro 33 .

Ethical approval and consent to participate
We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue.So, it is not applicable.
Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, must comply with relevant institutional, national, and international guidelines and legislation.
We confirmed that all methods followed the relevant guidelines/regulations/legislation.The authors have complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.Seeds were purchased from a certified seed dealer, so no permission is required.1 and 2).

Morphology and structural analyses of biochar
SEM analysis (Fig. 8A) revealed that the sBC sample exhibited a porous and heterogeneous structure with various nanoparticles attached to the biochar surface.The EDX analysis (Fig. 8B) confirmed the presence of multivalent metal elements in the sBC sample, possibly in hydrobiotite minerals.XRD analysis (Fig. 8C) identified crystalline phases in the sBC sample.Peaks corresponding to carbon (e.g., graphene) and silica (quartz) were observed, consistent with the EDX results.Peaks associated with calcium compounds, such as calcite (CaCO 3 ), were also detected, indicating potential sites for phosphorous adsorption on the biochar surface.Furthermore, diffraction peaks within the 60°-65° range indicated the presence of kaolinite and hydrobiotite minerals.FTIR spectroscopy (Fig. 8D) provided insights into the functional groups present in the sBC sample.Peaks in the 1600-1650 cm −1 range were attributed to C-X bonds, while a sharp peak at 1115 cm −1 indicated the presence of C-O/C-N conjugates.Peaks near 1380 cm −1 and above 750 cm −1 suggested Si-O-Si vibrations and Si-O-Si asymmetric bending vibrations, respectively.A broad vibration centered at about 1615 cm −1 indicated C=C stretching due to conjugated carbon, while vibrations in the 2020-2070 cm −1 range represented X=C=Y bonding.Vibrations between 3200 and 3400 cm −1 indicated O-H bonding in the sBC sample.

Discussion
Salinity is one of the major abiotic environmental factors that adversely affect crop productivity 34 .Salinity stress dramatically decreased the root and stem dry matter compared to the control treatment because of the direct impacts of ion toxicity or the indirect effects of salty ions that cause soil/plant osmotic imbalance.This judgment agrees with 35 .As a result of the salt effect on the plasma membrane's electrical potential, which decreased both ion and water absorption, creating water stress 36 , when wheat plants exposed to saline conditions showed lower RWC and MSI values.The findings from the present study revealed that saline soil significantly hindered plant growth and reduced wheat's relative water content (RWC) in the absence of Si-BC application.Consistent with these numerous other studies have also observed that salinity treatment further impairs plant growth.This could be attributed to the excessive accumulation of sodium, which disrupts water balance, restricts photosynthesis, and damages cell membranes 2,4,5,37 .
The plant was grown in Si-BC amended soil with NaCl treatment, which showed decreased salt stress and boosted plant height, leaf count, and dried fresh weight of the root and shoots.When wheat plants were exposed to 200 mM NaCl salt stress, the Si-BC applied at 1% and 2.5% positively affected growth traits, chlorophyll, leaf fluorescence, and nutrient concentration in above and below-ground (shoot and root) parts of the plant.Previous studies have also revealed the efficacy of biochar in reducing the salinity effects on wheat, sorghum, and maize crops [38][39][40] .The current study's findings showed that Si-BC increased wheat plant growth and biomass under salt stress by reducing the negative effects of salt stress.Incorporating doped biochar (SBC) significantly enhanced both the plant growth and grain yield of quinoa compared to undoped biochar.This outcome aligns with observations made by researchers in previous studies 41 , who demonstrated that biochar supplemented with silicon (Si) outperformed plain biochar in addressing salt stress.The increased growth observed in response to silicon under salt stress can be attributed to regulating antioxidant enzymes, enhanced nutrient uptake, and modulation of soil pH.Physiological indicators, including chlorophyll content and fluorescence parameters, decreased when plants were subjected to salinity stress.However, adding Si-BC mitigated these declines and improved these physiological attributes.These findings are consistent with earlier research demonstrating that biochar supplementation enhances these traits across various plant species facing salt stress 37 .Our results demonstrated that applying Si-BC caused levels of soluble sugar to increase in salt stress.Sugar plays a role in oxidative stress to eliminate ROS and is a vital component of membranes 42 .Their increased abundance during stressful settings is the breakdown of bigger carbohydrate molecules that keep the cell turgid 43 .
According to recent findings, salinity treatment caused significantly higher levels of H 2 O 2 and MDA than control plants.Increased levels of H 2 O 2 and MDA in wheat plants were the signs of oxidative stress 37 .Consistent with these findings, salinity induced oxidative stress and membrane damage in quinoa plants 37 .The inclusion of Si-BC alleviated salinity stress in the plants.The levels of H 2 O 2 and MDA were reduced, leading to improved stability of cell membranes in the presence of Si-BC.The detoxification of reactive oxygen species (ROS) is facilitated by various antioxidant enzymes within plant organelles 2,17,37 .Antioxidant enzymes are overproduced in the current study under salt stress to lower the levels of ROS, which supports the results of 37,44 .It was discovered that SOD activity increased when exposed to salt stress.Surprisingly, the addition of Si-BC under salinity increased antioxidant enzymes.Different studies have also reported the positive role of silicon nanoparticles doped biochar in increasing the antioxidant activities in plants growing on soil contaminated with NaCl salt 2,17,37 .
The introduction of salinity elevated the sodium (Na) concentration in wheat.Na ions tend to be sequestered in the vacuole rather than expelled by roots.This occurs because, under salinity stress, Na enters plant cells through potassium (K) channels. 45.Salinity also reduced the uptake of K in quinoa tissues 2,37 .The results of this study highlight the positive impact of Si-BC, which mitigates the accumulation of Na and enhances the uptake of K by wheat plants.Consequently, biochar emerges as an effective strategy for mitigating the adverse effects of salinity on plants by reducing the uptake of toxic ions while increasing the absorption of essential plant nutrients 5,37 .Silicon nanoparticle-based biochar proved even more effective in limiting the accumulation of toxic Na ions and promoting the uptake of essential K ions by wheat.It is well established that Si-BC enhances the uptake of nitrate and chloride due to improved soil nutrient status and facilitates root penetration.Moreover,    the presence of various compounds such as magnesium (Mg), calcium (Ca), and phosphorus (P) on the surface of Si-BC enhances cation exchange and water-holding capacity of the soil 46,47 .Our findings indicate that under salt stress, micronutrient uptake decreases.In contrast, biochar supplementation significantly benefits soil health and plant growth by providing essential elements such as iron (Fe), zinc (Zn), and manganese (Mn).

Conclusion
This is the first study of silicon nanoparticle-based biochar for reducing salinity-induced phytotoxicity in wheat.
The current study showed that adding Si-BC to salt-affected soil considerably improved its physicochemical properties, enhancing the physiology and overall growth of T. aestivum L. This may be ascribed to improved plant growth, increased water retention, improved nutrient supply, and increased stress tolerance.However, the results were visible when 2.5% Si-BC2 was applied under salt stress.Thus, it was shown that abiotic stresses (such as salinity stress) in the environment could be effectively tolerated by applying various rates of Si-BC; additionally, the adsorption efficiency could be doubled by optimizing it with the application of various types of biochar to improve soil fertility and crop yield.The recent study contributes new information about Si-BC ability to promote plant development in saline soils.

Figure 1 .
Figure 1.Impact of SiNP-based Biochar different application rates (1% and 2.5%) on chlorophyll a (A), chlorophyll b (B), total chlorophyll (C) and carotenoid (D) in wheat leaves under no stress and 200 mM NaCl stress.Data present the mean ± standard deviation of three replicates.Different letters on bars indicate significant differences (p ≤ 0.05) compared by Tukey's Test.

Figure 2 .
Figure 2. Impact of SiNP-based Biochar different application rates (1% and 2.5%) on Phi-2 (A), NPQt (B), Fv/Fm (C), and PhiNO (D) of wheat plant under no stress and 200 mM NaCl stress.Data presents the mean ± standard deviation of three replicates.Different letters on bars indicate significant differences (p ≤ 0.05) compared by Tukey's Test.

Figure 3 .
Figure 3. Impact of SiNP-based Biochar different application rates (1% and 2.5%) on SOD (A), POD (B), CAT (C), and APX (D) in wheat under no stress and 200 mM NaCl stress.Data presents the mean ± standard deviation of three replicates.Different letters on bars indicate significant differences (p ≤ 0.05) compared by Tukey's Test.

Figure 4 .
Figure 4. Impact of SiNP-based Biochar different application rates (1% and 2.5%) on H 2 O 2 (A), MDA (B), electrolyte leakage (C), relative water contents (D) in wheat plant under no stress and 200 mM NaCl stress.Data presents the mean ± standard deviation of three replicates.Different letters on bars indicate significant differences (p ≤ 0.05) compared by Tukey's Test.

Figure 5 .
Figure 5. Impact of SiNP-based Biochar different application rates (1% and 2.5%) on shoot Na (A), K (B), NO 3 (C) and Cl (D) concentration of wheat under no stress and 200 mM NaCl stress.Data present the mean ± standard deviation of three replicates.Different letters on bars indicate significant differences (p ≤ 0.05) compared by Tukey's Test.

Figure 6 .
Figure 6.Impact of SiNP-based Biochar different application rates (1% and 2.5%) on root Na (A), K (B), NO 3 (C) and Cl (D) concentration of wheat under no stress and 200 mM NaCl stress.Data present the mean ± standard deviation of three replicates.Different letters on bars indicate significant differences (p ≤ 0.05) compared by Tukey's Test.

Figure 7 .
Figure 7. Impact of SiNP-based Biochar different application rates (1% and 2.5%) on soil Na (A), K (B), NO 3 (C) and Cl (D) content under no stress and 200 mM NaCl stress.Data present the mean ± standard deviation of three replicates.Different letters on bars indicate significant differences (p ≤ 0.05) compared by Tukey's Test.

Figure 8 .
Figure 8. SEM image showing small macropores on the surface of sieved biochar (sBC) with granular features (A).EDX map clearly showing traces of various elements in the sample (B).The XRD pattern of sBC (C).Corresponding FTIR spectrum showing different functional groups present in sBC sample (D).
. However, an atomic

Table 1 .
Impact of Biochar 1% and 2.5% on the root length, shoot length, root dry weight, shoot dry weight of wheat plant under 200 mM NaCl stress.Data present the mean ± standard deviation of three replicates.

Table 3 .
Impact of Biochar 1% and 2.5% on the Number of spikelet's, Spike length (cm), spiked (g) and spike fresh weight (g)of wheat plant under 200 mM NaCl stress.Data present the mean ± standard deviation of three replicates.